Cultivation of novel Atribacterota from oil well provides new insight into their diversity, ecology, and evolution in anoxic, carbon-rich environments

Background The Atribacterota are widely distributed in the subsurface biosphere. Recently, the first Atribacterota isolate was described and the number of Atribacterota genome sequences retrieved from environmental samples has increased significantly; however, their diversity, physiology, ecology, and evolution remain poorly understood. Results We report the isolation of the second member of Atribacterota, Thermatribacter velox gen. nov., sp. nov., within a new family Thermatribacteraceae fam. nov., and the short-term laboratory cultivation of a member of the JS1 lineage, Phoenicimicrobium oleiphilum HX-OS.bin.34TS, both from a terrestrial oil reservoir. Physiological and metatranscriptomics analyses showed that Thermatribacter velox B11T and Phoenicimicrobium oleiphilum HX-OS.bin.34TS ferment sugars and n-alkanes, respectively, producing H2, CO2, and acetate as common products. Comparative genomics showed that all members of the Atribacterota lack a complete Wood-Ljungdahl Pathway (WLP), but that the Reductive Glycine Pathway (RGP) is widespread, indicating that the RGP, rather than WLP, is a central hub in Atribacterota metabolism. Ancestral character state reconstructions and phylogenetic analyses showed that key genes encoding the RGP (fdhA, fhs, folD, glyA, gcvT, gcvPAB, pdhD) and other central functions were gained independently in the two classes, Atribacteria (OP9) and Phoenicimicrobiia (JS1), after which they were inherited vertically; these genes included fumarate-adding enzymes (faeA; Phoenicimicrobiia only), the CODH/ACS complex (acsABCDE), and diverse hydrogenases (NiFe group 3b, 4b and FeFe group A3, C). Finally, we present genome-resolved community metabolic models showing the central roles of Atribacteria (OP9) and Phoenicimicrobiia (JS1) in acetate- and hydrocarbon-rich environments. Conclusion Our findings expand the knowledge of the diversity, physiology, ecology, and evolution of the phylum Atribacterota. This study is a starting point for promoting more incisive studies of their syntrophic biology and may guide the rational design of strategies to cultivate them in the laboratory. Video Abstract Supplementary Information The online version contains supplementary material available at 10.1186/s40168-024-01836-7.


Background
Members of the phylum Atribacterota are found in a broad range of anoxic environments, including oil reservoirs [1] and produced water [2], hot springs [3], marine sediments [4], and methane hydrates [5].Currently, only one species, Atribacter laminatus RT761 T , has been isolated and characterized [2]. A. laminatus, a glucose fermenter, produced H 2 , acetate, and CO 2 as fermentation products and was stimulated by cocultivation with a hydrogenotrophic methanogen [2].This metabolism, along with the previous enrichment of two Candidatus Caldatribacterium species on lignocellulose suggested widespread sugar fermentations within the class Atribacteria, known informally as OP9 [6].However, this function is not well understood due to poor cultivability and a paucity of high-quality genomes of Atribacterota.Interpretations of additional single-amplified genomes (SAGs) and metagenomeassembled genomes (MAGs) and their environmental origins have also hinted at anaerobic hydrocarbon fermentation and either syntrophic acetate oxidation or homoacetogenesis via the Wood-Ljungdahl pathway (WLP) [1][2][3][4][5]7].Although these studies provide valuable insights into these organisms, the presence of only a single pure culture and the scarcity of experimental studies targeting Atribacterota obscure a more comprehensive understanding of the phylum's nature.
In this study, we describe the isolation of a second pure culture of Atribacterota, herein named Thermatribacter velox B11 T , and hydrocarbon-degrading enrichments containing a member of the JS1 class, herein named Phoenicimicrobium oleiphilum HX-OS.bin.34 TS .We also probe the core metabolism of Atribacterota, which revealed the importance of the reductive glycine pathway (RGP), and not the WLP, in the core metabolism of Atribacterota.We suggest a central metabolic core of the RGP and diverse hydrogenases, with the variable presence of glycoside hydrolases, fumarate-adding enzymes (Fae), CO dehydrogenase/ acetyl-CoA synthase (AcsABCDE), and formate dehydrogenase (FdhAB) endow Atribacterota with different roles in syntrophic networks in a variety of anoxic, carbon-rich environments.

Sample collection and acetate enrichment
Crude oil sludge samples were collected from the Haoxian Oil Reservoir (HX-OS) that originated between 1000 and 2000 m deep with a temperature range of 60-80 °C [8], which is part of the Shengli Oilfield located in Shandong Province, China (E 118°31′30′′, N 37°24′00′′) in April 2017.Anaerobic enrichment cultures were set up (Figure S1), initially using a pre-reduced medium (PRM) at 75 °C as described previously [8].Following incubation for 300 days, 10 ml of culture was inoculated into 20 ml of PRM supplemented with 10 mM acetate (culture HX-AS) and incubated at 75 °C.After 100 days, culture HX-AS was subcultured and transferred at an interval of approximately 30 days by inoculating 20 ml cultures into 600 ml serum bottles containing 20 ml PRM supplemented with 10 mM acetate to enrich for an acetatedegrading methanogenic consortium (Figure S2).Acetate consumption and methane production were detected using high-performance liquid chromatography (HPLC, LC 1200, Agilent) and gas chromatography (GC 7820A, Agilent) as described previously [8].

Isolation and complete genome sequencing of B11
Previous genomic studies have suggested syntrophic acetate oxidation in Atribacterota [4,9,10].Therefore, we targeted acetate-degrading Atribacterota from the enrichment culture HX-AS [11] using the anaerobic rolltube method to obtain colonies [12,13] by using a lowsalt (LS) medium supplemented with 10 mM sodium acetate.The LS medium contained (per liter) 9 g NaCl, 3 g MgCl 2 •6H 2 O, 0.15 g CaCl 2 •2H 2 O, 0.3 g NH 4 Cl, 0.2 g KH 2 PO 4 , 0.5 g KCl, 7.16 g HEPES, 2 ml trace elements solution, 2 ml vitamins solution [14], 2.52 g NaHCO 3 , 0.5 g Na 2 S•9H 2 O, and 0.5 ml resazurin solution (1 g/L).Ten millimeter sodium acetate and 15 g/L Gelzan TM CM were used as substrate and solidifying agents, respectively.The medium was prepared under a gas mixture of N 2 :CO 2 (80:20) using modified Hungate anaerobic techniques [15].Vitamins, NaHCO 3, and Na 2 S•9H 2 O were autoclaved separately from sterile anaerobic solutions at 121 °C for 30 min and added to the medium.The final pH of the medium was adjusted to 6.8-7.0.Approximately 0.5 ml enrichment cultures were inoculated into Hungate tubes containing LS medium supplemented with 10 mM sodium acetate after a series of dilutions (10 −1 to 10 −8 ).All tubes were incubated at 75 °C.Following incubation for 2 to 4 weeks, single colonies were picked and transferred into the liquid isolation medium.The purified isolates were identified by calculating sequence similarities of partial 16S rRNA gene sequences using NCBI Blast.DNA extraction, 16S rRNA gene amplification, and sequencing were performed according to methods described previously [16] using the primer set 27F/1492R [16,17].Subsequently, the colonies identified as members of the Atribacteria were purified by repeated restreaking onto roll-tubes.A single colony designated as B11 T was obtained.The purity of strain B11 T was confirmed by microscopic observation and repeated sequencing of the 16S rRNA gene (OQ519918).As strain B11 T grew slowly in LS medium with acetate as the sole carbon source, a nutrient-rich LS medium containing 10 mM glucose and 0.5 g/L yeast extract was used for subsequent culturing.For whole genome sequencing, genomic DNA of strain B11 T was extracted from cells harvested at the exponential phase using the ZYMO HMW gDNA kit (ZYMO, California, USA) according to the manufacturer's instructions.The whole genome was sequenced using Illumina NovaSeq PE150 at the Beijing Novogene Bioinformatics Technology Co., Ltd.Genome assembly was performed using SOAPdenovo (version 2.04) [18], SPAdes [19] and ABySS [20], followed by CISA [21] to integrate the assemblies.Gaps were filled with gapclose (version 1.12), thereby generating a closed, circular genome (CP121689).Default parameters were used for all the assembly and gap closure steps.

Microscopic observation
Cultures of strain B11 T grown in the nutrient-rich LS medium at 75 °C for 48 h were used to determine cell morphology and structure.Gram staining was performed using a Gram-staining kit (Solarbio, China) and was observed with microscopy (Eclipse 80i, Nikon).Cell shape and size were observed with scanning electron microscopy (EVO18, Zeiss).Samples washed with sodium phosphate buffer (PBS) (0.01 M, pH 7.2) were fixed with 2.5% glutaraldehyde at 4 °C for 12 h, washed thrice with PBS, and dehydrated in a series of ethanol and with tert-butyl alcohol for 15 min each.The sample was then kept at −80 °C for 12 h, freeze-dried in a lyophilizer (FreeZone ® 6, Labconco), and finally coated with gold before observation.Cell structures were examined by transmission electron microscopes (TEMs) using negative staining and ultramicrotomy.To prepare for negative staining, 2 μl of active cultures were dropped on formvar film-coated copper grids (200 mesh) and dried at room temperature.Samples were stained for 1 min with neutral 1% (w/v) phosphotungstic acid solution, excess reagent washed with ultrapure water, air-dried, and observed in a field emission transmission electron microscopy (Tecnai G2 F20 S-TWIN, FEI).To prepare thin slices for TEM observation, cells were prefixed with 3% glutaraldehyde at 4 °C for 12 h and then postfixed in 1% osmium tetroxide at 4 °C for 2 h, dehydrated in a gradient series of acetone, infiltrated and embedded in Epon 812.The semi-thin sections were stained with methylene blue.Ultrathin sections were cut with an ultramicrotome (EM UC7, Leica), and stained with uranyl acetate and lead citrate.Sections were examined with a TEM (JEM-1400-FLASH, JEOL).

Physiological characterization
Physiological tests for strain B11 T including temperature, pH, and potential fermentation substrates were performed in the nutrient-rich LS medium containing 10 mM glucose and 0.5 g/L yeast extract unless otherwise indicated.The NaCl concentration range was tested in a NaCl-free medium.Substrate utilization was determined in LS medium supplemented with 0.2 g/L yeast extract and potential fermentation substrates; LS medium supplemented with 0.2 g/L yeast extract was used as control.Growth of strain B11 T was determined by monitoring the optical density at 600 nm (OD 600 ) with a cell density meter (Ultrospec 10, biochrom).Metabolites in the gaseous phase (hydrogen and carbon dioxide) were measured with gas chromatography (GC) equipped with a thermal conductivity detector (GC 2010, Shimadzu) and a Porapak Q column.Ethanol in the liquid phase was measured using a GC (GC 7890A, Agilent) equipped with a flame ionization detector and DB-WAX column (length, 30 m; inner diameter, 0.32 mm), while lactate, formate, acetate, propionate, butyrate, and iso-valerate were measured using a HPLC (LC 1200, Agilent) equipped with Aminex HPX-87H column (Bio-Rad).
Oxygen tolerance was tested in Hungate anaerobic tubes containing the anaerobic nutrient-rich LS medium which contains 10 mM glucose and 0.5 g/L yeast extract, but without reducing agents (Na 2 S or cysteine).Different volumes of oxygen were added into the headspace of the tube (0, 1, 2, and 5%).OD 600 was measured to determine the growth of the isolate.

Chemotaxonomic analyses
Cells incubated in the nutrient-rich LS medium at 70 °C for 48 h were used to analyze cellular fatty acids, polar lipids, and respiratory quinones.The methyl esters of fatty acids were prepared and extracted from freezedried biomass according to the protocol described by Eder [22].The esterified fatty acids were separated with a gas chromatograph-mass spectrometer (8860 GC system with 5977B GC/MSD, Agilent), and identified with Sherlock Microbial Identification System (MIDI Inc, Newark).Polar lipids and respiratory quinones were extracted following a previously published protocol [23].The purified respiratory quinones were analyzed with HPLC (1260 Infinity II, Agilent) [24].

DNA/RNA extraction and next generation sequencing
DNA for metagenomic sequencing was collected from 10 to 15 g of oil sludge (HX-OS) or from culture HX-AS, and DNA was extracted using bead-beating methods [25].DNA libraries were generated by Novogene using a TruSeq DNA PCR-Free kit for Illumina (CA, USA) according to the manufacturer's instructions.
For metatranscriptomic sequencing, cells were collected from about 15-20 ml of the sample from the oil reservoir (HX-OS) by centrifugation (17,000×g, 5 min, 4 °C; Beckman Coulter), transferred immediately to liquid nitrogen, and stored at −80 °C.Cells were lysed by using the bead-beating method (Fast-Prep-24, MP), and total RNA was extracted using an acid phenol:chloroform:isoamyl alcohol method [26].Quality checking, genomic DNA digestion, ribosomal RNA removal, cDNA synthesis, and library construction were performed by Novogene.Metagenomic and metatranscriptomic sequencing were both performed using the NovaSeq 6000 instrument with PE150 at Novogene.

Metagenomic and metatranscriptomic analyses
The raw metagenomic reads were quality filtered by using fastp [27] with the parameters (-q 20 -u 20 -e 20 -l 50).High-quality reads were de novo assembled using SPAdes [19] with parameters --meta -k 21,33,55,77,99.Genome binning was performed by using MetaBAT [28] as previously described [29], based on scaffolds with lengths > 2.5 kbp.The completeness and contamination of each MAG were calculated using CheckM [30].MAGs with estimated completeness < 50% or contamination > 10% were discarded from this study as low-quality MAGs per MIMAG standards [31].Medium-and high-quality MAGs were grouped into species clusters based on 95% average nucleotide identity (ANI) values determined by pyANI [32].The genomes with the highest quality in each species cluster were selected as cluster representatives.A genome database comprised of the 33 representative genomes was then constructed and used for genome annotation and community function reconstruction.The Genome Taxonomy Database (GTDB)-Tk [33] was used to assign a taxonomy to the representative genomes by using relative evolutionary divergence (RED).The iRep (index of replication) [34] analysis was used to estimate the replication rate of each species in situ.Briefly, highquality reads from each metagenome were mapped to the representative genomes by using Bowtie2 [35], and the read mapping data was then used for calculating the iRep values with default parameters.The relative abundance of taxa in HX-OS and HX-AS was examined by Kaiju software [36] with high-quality reads.Raw metatranscriptomic data were pre-processed as described for metagenomic data.The rRNA sequences from the resulting high-quality reads were removed by SortMeRNA [37] (--fastx --sam --aligned --other --paired_in --out2 -e 5 --de_novo_otu --otu_map).Finally, the filtered metatranscriptomic reads were mapped to the Atribacterota genes by BBmap v. 38.85 with a sequence similarity threshold of 95% (http:// sourc eforge.net/ proje cts/ bbmap/).

Phylogenetic analysis
A set of 48 Atribacterota genomes (Table S1) included new genomes from this study and high-and mediumquality species references from the Genome Taxonomy Database (completeness > 80% and contamination < 5%).The 16 ribosomal proteins were extracted and identified by using AMPHORA2 [38].All marker proteins were aligned using the MUSCLE program [39] with 100 iterations.The TrimAL program [40] (-gt 0.95 -cons 50 -htmlout) was used to eliminate the poorly aligned regions, and finally, 2393 amino acid positions were kept for further analysis.The alignments were concatenated by using the Perl script (https:// github.com/ nylan der/ catfa sta2p hyml).A maximum-likelihood phylogeny was calculated by IQ-Tree [41] with parameters (-alrt 1000 -bb 1000 -nt AUTO).The best-fit model (LG+F+R5) is well supported by the Bayesian Information Criterion (BIC).
The MrBayes [43] was used to construct the Bayesian tree with parameters (ngen = 1,000,000 Nruns = 2 Nchains = 4 diagnfreq = 1000 relburnin = yes burninfrac = 0.25 samplefreq=100 printfreq = 100) using the multiple sequence alignment of the concatenated ribosomal proteins.The results of the standard deviation of split frequencies (< 0.01), the potential scale reduction factor (PSRF = 1), and the effective sample size (ESS > 100) made the Bayesian tree highly reliable.The time-calibrated phylogenetic tree was further inferred by using R with the ape package.All trees were visualized and annotated by using iTOL.

Comparative genomic analysis
The protein-coding sequences (CDSs) of each genome were predicted by using Prodigal [44] with the parameter "-p single", and the CDSs were then annotated against egg-NOG and KEGG databases by using DIA-MOND [45] with E-values < 1e-10.The predicted CDSs were also uploaded to the KEGG Automatic Annotation Server [46] with "for prokaryotes" and "bidirectional best hit" options for further function confirmation and metabolic pathway reconstruction.Average amino acid identity (AAI) values of each pair of genomes were calculated as described earlier [29].The orthologous protein sequences were identified based on reciprocal best BLAST hits, and the mean identity of all orthologs was calculated as the AAI value.The 95% ANI value was used as the threshold for defining species [47].The rRNAs and tRNAs were predicted by using RNAmmer [48] and tRNAscan-SE [49], respectively.Clusters of orthologous genes were generated by KEGG based on representative genomes.The evolutionary history of Atribacterota was inferred by using COUNT [50] as previously described [29,51,52].Briefly, the gain-lossduplication model with the Poisson family size distribution at the root was used for the calculation of the rate models.The rate variation across families was evenly distributed into four gamma categories: edge length, loss rate, gain rate, and duplication rate, each set at a 1:1:1:1 ratio.Convergence criteria were established at 100 rounds for maximum number of optimization rounds, with a 0.1 convergence threshold on the likelihood.Ancestral inference and family dynamics analyses were performed with the option Dollo parsimony.

Laboratory enrichments of Phoenicimicrobium oleiphilum and metagenomic/metatranscriptomic analysis
Cultivation strategies were guided by multi-omics data [25] to test the degradation of n-alkanes in Atribacterota.The laboratory enrichments of oil sludge were performed in anoxic PRM at 55 °C with a variety of substrates, including 1 ml/L hexadecane, 2 ml/L eicosane, 0.6 g/L triacontane, and additional 200 g/L oil sludge, respectively.After 1 month of cultivation, 15-20 ml of each enrichment culture was collected, and DNA/ RNA was extracted.Metagenomic/metatranscriptomic sequencing and analysis were performed as described above.

Development of a long-term acetate-oxidizing culture containing Atribacterota from a crude oil reservoir
Oil sludge (HX-OS) that originated from 1000 and 2000 m deep within the Shengli Oilfield, China, was initially collected to study anaerobic acetate oxidation.Anaerobic enrichment cultures were continuously passaged at 75 ºC with 10 mM acetate as the sole source of carbon for nearly 4 years (culture HX-AS; Figure S1), after which amendments of 3 mM acetate were consumed reproducibly within 30 days, with the concomitant production of ~ 2.5 mM CH 4 (Figure S2), implying acetoclastic methanogenesis or syntrophic acetate oxidation.To identify the microbial community members within the HX-AS enrichment culture and the original sample from the Haoxian Oil Reservoir (HX-OS), metagenomic assemblies were taxonomically assigned (Fig. 1a; Table S2), binned, and annotated, resulting in 33 representative high-quality MAGs based on 95% average nucleotide identity (ANI) (Table S3).The predominant archaea belonged to the Euryarchaeota (GTDB_id: p__Halobacteriota and p__Methanobacteriota) and contained mcrABCDG genes, suggesting they are involved in methane metabolism [53].The predominant bacteria belonged to the Bacillota (Firmicutes), particularly classes Moorelia and Thermoanaerobacteria.These MAGs encode the Wood-Ljungdahl pathway, which is consistent with their potential roles in syntrophic acetate oxidation in the acetate-enriched culture [54] (Table S3).
Two MAGs (HX-AS.bin.3 and HX-OS.bin.34) were assigned to the classes Atribacteria (OP9) and JS1 within the phylum Atribacterota according to the GTDB-Tk [33].The presence of HX-AS.bin.3, a member of class Atribacteria, in the long-term acetate-amended enrichment (0.31 % in HX-AS; Table S3) is consistent with a previously proposed role of some Atribacterota in syntrophic acetate oxidation [1][2][3][4][5]7].However, genes coding for the Wood-Ljungdahl pathway (acsABCED) were not detected in HX-OS.bin.34 and HX-AS.bin.3, which obscured their functions in these environments.The detection of faeABCDEFJ genes in HX-OS.bin.34 is consistent with the inferred genomic ability of some members of the JS1 group to ferment short-chain n-alkanes into fatty acids [1], and aligned with the relatively high abundance of this species in the oil reservoir sample (2.33% in HX-OS; Table S3).

Laboratory isolation of the second member of Atribacterota from an acetate-amended enrichment
To evaluate the potential role of HX-AS.bin.3 in the acetate-amended enrichment, anaerobic isolation was performed by using the acetate enrichment culture HX-AS as the inoculum.The isolate B11 T was obtained on a low salt (LS) medium supplemented with 10 mM acetate and represents the second pure culture in the phylum Atribacterota (Fig. 1b-d; Supplementary information; Table S4).Although strain B11 T was isolated on LS medium supplemented with acetate as the only source of carbon, B11 T could not be maintained on the same medium.Correspondingly, the key genes encoding the CO dehydrogenase/acetyl-CoA synthase complex (acsABCDE) and formate dehydrogenase (fdhAB) for the WLP were absent in the complete genome of B11 T , which makes syntrophic acetate oxidation highly improbable.Further experiments showed that B11 T was unable to grow with acetate, or glucose alone, but it could be maintained on a LS medium supplemented with 10 mM glucose and 0.5 g/L yeast extract (Figure S3a).Therefore, LS medium with 10 mM glucose and 0.5 g/L yeast extract was used as the basic culture medium to carry out physiological experiments.The experiments showed that isolate B11 T is anaerobic but was able to tolerate up to 2% oxygen (Figure S3b), and the genome analysis results indicated that it contains multiple genes for detoxification of reactive oxygen species (Table S6: peroxidase, alkyl hydroperoxide reductase, alkylhydroperoxidase, and superoxide reductase).It has an optimum growth temperature of 70 ºC (Figure S3c), optimal pH of 7.0 (Figure S3d), and optimal NaCl concentration of 10 g/L (Figure S3e).These traits are consistent with its environmental source in the deep subsurface within high-temperature brines associated ).The maximum-likelihood phylogenetic tree was constructed using the concatenated alignment of 16 ribosomal proteins identified by using AMPHORA2 [38,65].All marker genes were aligned using the MUSCLE program [39] with 100 iterations, respectively.The TrimAL software [40] was used to eliminate the poorly aligned regions, and finally, 2393 amino acid positions were kept for further analysis.The alignments were concatenated and then used for calculating a maximum-likelihood phylogeny by IQ-Tree [41].The best-fit model LG+F+R5 is well supported by the Bayesian Information Criterion.Bootstrap values were based on 1000 replicates and nodes with percentages > 80% were indicated as black circles.The red circles listed on the right illustrate the genome sources.The lineages with red stars showed the phylogenetic placement of novel lineages from this study with the Haoxian Oil Reservoir.Average nucleotide identity (ANI) analysis showed that the complete genome of isolate B11 T and MAG HX-AS.bin.3 from the acetate enrichment are the same species (98%) by using 95% ANI as the cut-off [47] (Figure S4).Therefore, the genome of strain B11 T and MAG HX-OS.bin.34,together with 46 high-quality Atribacterota genomes from other sources (Table S5), were subjected to further analyses to assess their diversity, physiology, and evolution, especially in acetate-and hydrocarbon-enriched environments.

Phylogenomic analyses reveal two different Atribacterota in the Haoxian Oil Reservoir of Shengli Oilfield and support the division of Atribacterota into two major classes
Phylogenetic analysis confirmed that these 48 genomes separated into the two known major lineages of Atribacterota (Fig. 1e), the class Atribacteria (OP9) and the class informally named JS1 [2,3].Isolate B11 T branched from a basal node within the Atribacteria as a sister group to the family Ca.Caldatribacteriaceae.The phylogenetic position and distinctness of B11 T based on relative evolutionary divergence indicated that B11 T is a novel species of a new family within the order Atribacterales (Fig. 1e; Table S5).Here, we propose the name Thermatribacter velox B11 T as the first pure culture of the novel family Thermatribacteraceae.In contrast, MAG HX-OS.bin.34,derived from the natural oil sample, belongs to the class previously defined as JS1 [3].We propose to name JS1 herein under the SeqCode as Phoenicimicrobiia, with the nomenclatural type being Phoenicimicrobium oleiphilum HX-OS.bin.34TS .AAI and ANI analyses were used in conjunction with the phylogenomic analysis to further understand the diversity of existing Atribacterota genomes (Supplementary information and Figure S5-S6).Our refined taxonomic classification resulted in 2 classes, 2 orders, 5 families, 13 genera, and 31 species represented by high-quality MAGs and complete genomes of isolates, which significantly expands the previously published three species in the Atribacterota (Atribacter laminatus RT761 T , Ca. Caldatribacterium saccharofermentans, and Ca.Caldatribacterium californiense).Taxonomic names are proposed within protologues at the end of the main text and in Table S5.

Genomic properties and functional annotation of Atribacterota genomes
Atribacterota genome sizes ranged from 1.2 Mb (Gw_SlDig_bin_137) to 3.1 Mb (Atribacter laminatus RT761 T ), with smaller genome sizes in the Atribacteria than in Phoenicimicrobiia (Wilcoxon, p < 0.05; Figure S7; Table S1).Principal coordinates analysis (PCoA) based on the KEGG orthologs (KOs) and Clusters of Orthologous Groups (COGs) showed that genomes clustered according to their taxonomy (Figure S8), underscoring the functional divergence of the two classes.Metabolic pathways inferred from the genomes revealed conserved central carbon metabolic pathways, including complete pathways for glycolysis, gluconeogenesis, pentose phosphate pathway, and the incomplete horseshoe-type tricarboxylic cycle, which is used to generate anabolic intermediates (Fig. 2; Table S6).Considering the absence of cytochrome c/bd oxidase and superoxide reductase in all genomes (Table S6), the Atribacterota are probably all strict anaerobes.COG categories that were significantly different between Atribacteria and Phoenicimicrobiia included those for motility (category N), carbohydrate transport and metabolism (category G), coenzyme transport and metabolism (category H), and signal transduction mechanisms (category T) (Figure S9).Genes for flagellum assembly and chemotaxis (category N) were found in all 15 genomes of the families Thermatribacteraceae (F1) and Ca.Caldatribacteriaceae (F2) within class Atribacteria, consistent with previous genomic predictions [6]; however, no flagellar components were detected in B11 T (Fig. 1b-d) and Atribacter laminatus RT761 T [2].The enrichment of genes for carbohydrate transport and metabolism (category G) in Atribacteria is consistent with sugar catabolism by B11 T and Atribacter laminatus RT761 T and suggests the two classes of Atribacterota have different carbon metabolisms.Genes encoding bacterial microcompartments in Atribacteria (OP9) are significantly more widespread than in Phoenicimicrobiia (JS1).Phoenicimicrobiia only has two genomes (RBG_19FT_COMBO_35_14 and Bin1_14.8m)simultaneously containing COG4576 and COG4577 (Table S6).Furthermore, putative selenate reductase gene ygfK was only detected in Phoenicimicrobiia, indicating the potential serenate reduction coupled with respiration for energy metabolism.These results also imply functional differences between the two classes.

Conserved carbohydrate fermentation in Atribacteria (OP9)
Analysis of the first high-quality Atribacteria genomes from lignocellulose enrichments suggested sugar fermentation, similar to the known physiology of A. laminatus RT761 T , which ferments glucose, producing H 2 , acetate, CO 2 , and trace amounts of ethanol [3,6].The isolation of B11 T allowed additional experimentation on a member of a new family of Atribacteria.Considering the physiology of A. laminatus RT761 T and the presence of genes encoding NADP-reducing NiFe (Group 3b) hydrogenases, FeFe (Group A3 and C) hydrogenases (Figure S10-12), acetogenesis (pta, ackA, acyP, eutE, adhP, and yiaY), alcohol dehydrogenase (adhP, and yiaY), and Rnf complex (rnfABCDEG), we speculated that B11 T may ferment sugars to acetyl-CoA, which could then be further used for reoxidation of NADH via acetyl-CoA reduction to acetate, ethanol, and H 2 (Fig. 2).To assess this prediction, we tested the growth of strain B11 T (70 ºC and pH 7) in media supplemented with different carbohydrates (d-mannose, d-fructose, d-ribose, d-xylose, d-galactose,  d-lactose, d-trehalose, and xylan) adjusted to a final concentration of 10 mM (5 g/L for xylan) and checked for growth and the formation of formate, acetate, propionate, ethanol, H 2, and CO 2 .The results showed that d-mannose, d-fructose, d-ribose, d-xylose, d-galactose, and xylan can be utilized by B11 T , similar to Atribacter laminatus RT761 T (Table S7), and fermentation products included acetate, H 2 , and CO 2 (Fig. 3).These results support prior suggestions that sugar fermentation may be universal within the Atribacteria [2].We also found that the number of glycoside hydrolase (GH) families was significantly higher in Atribacteria genomes than in Phoenicimicrobiia genomes (Figure S13).GHs may be used for depolymerization of xylan and other oligo-and polysaccharides.The near-universal presence of genes encoding GHs, NiFe Group 3b hydrogenases, FeFe Group A3 hydrogenases, acetogenesis, and Rnf complexes suggests sugar fermentation to acetate, H 2 , and CO 2 is a common core metabolism in the class Atribacteria (Figure S14).

Anaerobic hydrocarbon degradation might be prevalent in Phoenicimicrobiia (JS1)
We next focused our attention on MAG HX-OS.bin.34, which belongs to the class Phoenicimicrobiia (JS1).Putative fumarate-adding enzymes (FAE) were detected in the MAG HX-OS.bin.34, which is in line with a previous report suggesting the capacity of some members of Phoenicimicrobiia (JS1) to ferment short-chain n-alkanes [1].However, this function in Phoenicimicrobiia has not been investigated experimentally.Fumarate addition to methyl groups via FAEs is a well-known mechanism to initiate anaerobic catabolism of hydrocarbons [55].Genes encoding putative FAEs were also found in MAGs from wastewater and represented four non-monophyletic genus-level groups herein designated as Oleincola (G8), Oleihabitans (G9), Phoenicimicrobium (G10) and Immundihabitans (G11), all belonging to the class Phoenicimicrobiia (Fig. 1e, Table S5).The broad phylogenetic distribution of FAE-encoding MAGs and their presence in both oil reservoirs and wastewater suggests broad importance for hydrocarbon metabolism in Phoenicimicrobiia (Figure S15).Phylogenetic analyses showed that the putative alpha-subunits of FAE (faeA) from the Phoenicimicrobiia MAGs form a single clade that branched deeply among members of the fumarate reductase superfamily, which includes protein sequences encoded by assA, bssA, nmsA, and hbsA genes (Fig. 4).The  4 Phylogenetic tree of Phoenicimicrobiia putative FaeA.Reference sequences of AssA, BssA, HbsA, and HmsA were obtained from previous studies [1].The genes were aligned using MUSCLE [39] with 100 iterations, and divergent regions were eliminated using TrimAL [40].The IQ-Tree was used for phylogenetic inference [41], and the best model LG+F+I+G4 was well supported by Bayesian Information Criterion (BIC).Bootstrap values > 80 were shown in black dots monophyly of faeA homologs from Phoenicimicrobiia MAGs and synteny of the faeABCDEF gene cluster [1] suggests these genes were likely present in the common ancestor of the class Phoenicimicrobiia and extended by vertical inheritance.The presence of faeABCDEF explains why Phoenicimicrobium oleiphilum HX-OS.bin.34TS was relatively abundant in the original sample from the Haoxian Oil Reservoir (Table S3).

Reductive glycine pathway rather than the Wood-Ljungdahl pathway supports formate/CO 2 assimilation and syntrophic acetate oxidation
The genetic capacity for syntrophic acetate oxidation or carbon fixation through the WLP was reported in Atribacterota genomes in previous studies [4,9,10].Almost all Atribacterota genomes possess genes for formatetetrahydrofolate ligase (fhs), methylenetetrahydrofolate dehydrogenase (folD), and methylenetetrahydrofolate reductase (metF), which together with fdhAB, constitutes the methyl-branch of the WLP.However, we note that only members of Phoenicimicrobiia (JS1) contain the fdhAB genes necessary for reducing CO 2 to formate.Furthermore, only two Atribacterota genomes possess genes encoding the CODH/ACS complex (acsA, acsB, acsC, acsD, and acsE), the key genes for the carbonyl branch of WLP.These two genomes belong to a single genus-level group in the class Atribacteria, herein designated Nitricultor (G5) (Atribacterota bacterium CSSed162cmB 266 and Atribacterota bacterium CSSed11 91).
To further investigate the evolutionary history of the Nitricultor CODH/ACS complex, we reconstructed phylogenetic trees according to previous studies [29,42].The phylogenetic trees showed that the Nitricultor CODH/ ACS complex proteins (acsABCDE) were nested within a set of homologs from the phylum 'Ca.Bipolaricaulota' , suggesting these genes arose from a single horizontal gene transfer event (Fig. 5; Figure S16-S17).Given the absence of key genes encoding the methyl-branch (fdhAB) in Atribacteria and key genes encoding the carbonyl-branch (acsABCDE) in most Atribacterota, members of this phylum appear to be incapable of reducing CO 2 or oxidizing acetate via the WLP.Considering experimental evidence that some Atribacterota became labeled after incubation of partially serpentinized rocks and sediments from the Mid-Atlantic Ridge with 13 C-bicarbonate and H 2 or with 13 C-acetate [7], we explored the possibility Fig. 5 Phylogenetic tree based on concatenated AcsDABCE sequences.Maximum likelihood phylogeny of concatenated AcsDABCE sequences was performed as previous studies [29,42].The subunits of AcsD, AcsA, AcsB, AcsC, and AcsE were aligned MUSCLE with 100 iterations [39], respectively.The alignments were concatenated by using a perl script (https:// github.com/ nylan der/ catfa sta2p hyml).IQ-Tree [41] was used for phylogenetic inference with parameters (-alrt 1000 -bb 1000 -nt AUTO).The best-fit model was LG+F+R8, which was well supported by the Akaike Information Criterion (AIC), Corrected Akaike Information Criterion (cAIC) and Bayesian Information Criterion (BIC).Phylogenetic tree was visualized and annotated using iTOL [66] that some Phoenicimicrobiia may have the genetic capacity to reduce CO 2 to formate using fdhAB.
The only known natural carbon-fixation pathway that can assimilate formate is the reductive glycine pathway (RGP) [56].Surprisingly, the key genes for the RGP (fhs, folD, glyA, gcvT, gcvPA, gcvPB, pdhD) as well as fdhAB were nearly universal in Phoenicimicrobiia and some Atribacteria genomes (which lack fdhAB), including isolate B11 T , indicating that the RGP is likely the route for CO 2 /formate assimilation.In reverse, these enzymes could facilitate their participation in syntrophic acetate oxidation, rather than the previously reported WLP [4,9,10].

Evolutionary history of syntrophic physiology in Atribacterota
Following ancestral gene content reconstruction and phylogenetic analyses, we examined the gain and loss of genes relevant to syntrophic functions throughout the evolutionary history of Atribacterota (Fig. 6).As noted previously, genes for the CODH/ACS complex were largely absent in Atribacterota and were only gained from 'Ca.Bipolaricaulota' via HGT to the single genus Nitricultor (Fig. 5; Figure S16-S17).In contrast, the RGP and diverse hydrogenases are prevalent in extant Atribacterota, and the ancestral character state reconstruction indicated that these genes were ancestral within the phylum.Thus, we interpret that hydrogenases and the RGP are a part of the core metabolism of the Atribacterota and were critical for its evolution.
To gain more insight into the evolutionary history of these genes within the phylum, the protein sequences of the RGP enzymes and hydrogenases that supply reducing power for the RGP were selected for phylogenetic analyses: NiFe hydrogenase group 1-3 (Figure S10), NiFe hydrogenase group 4 (Figure S11), FeFe hydrogenase (Figure S12), fdhA (Fig. S18), fhs (Figure S19), folD (Figure S20), glyA S21), gcvT (Figure S22), gcvPA (Figure S23), gcvPB (Figure S24), and pdhD (Figure S25).If RGP-based metabolism was present in the last common ancestor of Atribacterota, it is expected that their RGP core proteins would be monophyletic.However, the phylogenetic trees revealed two distinct evolutionary trajectories between the classes Atribacteria and Phoenicimicrobiia (Figure S10-12; Figure S18-S25).Our results strongly suggest that the two classes of Atribacterota obtained genes for the RGP, hydrogen metabolism, hydrocarbon degradation, and sugar fermentation independently through lateral gene transfer after their divergence (Fig. 6).Given this set of common core metabolic genes, but with different evolutionary histories, we developed genome-resolved models based on MAGs derived from microbial communities in the hydrocarbon-rich oil reservoir sample (HS-OX) and the acetate enrichment (HS-AX) to further understand potential ecological roles of Phoenicimicrobium and B11 T .

The ecological function of Phoenicimicrobium oleiphilum (JS1) in hydrocarbon-enriched environments
In the original sample from the Haoxian Oil Reservoir, the dominant member of the Atribacterota was Fig. 6 Evolutionary history of syntrophic function in Atribacterota.The Bayesian tree topology was determined by MrBayes [43] by using multiple sequence alignments of 16 ribosomal proteins.The results of the standard deviation of split frequencies (< 0.01), the potential scale reduction factor (PSRF = 1), and the effective sample size (ESS > 100) made the Bayesian tree highly reliable.The time-calibrating phylogenetic tree was further inferred by using R with the ape package.The evolutionary history of Atribacterota was inferred by using COUNT [50] as previously described [29,51], and further confirmed by phylogenetic trees of each protein set Phoenicimicrobium oleiphilum HX-OS.bin.34TS , with a relative metagenomic read abundance of 2.33% (Table S3).Metatranscriptomic reads were obtained directly from samples from the Haoxian Oil Reservoir mapping to the HX-OS.bin.34 genome were also relatively high at 1.32% (Table S8), suggesting it is highly active.Almost all the genes encoded by HX-OS.bin.34 were highly expressed (Table S8), including the genes for FAE, RGP, acetogenesis, and hydrogenases, which is consistent with the inferred metabolism of alkane degradation.The role of Phoenicimicrobium oleiphilum in n-alkanes degradation was further confirmed by establishing short-term anaerobic enrichment cultures with samples from the Shengli Oilfield that were amended with crude oil, hexadecane, eicosane, or triacontane (Fig. 7; Table S9-10).MAGs with > 98% ANI values to Phoenicimicrobium oleiphilum HX-OS.bin.34TS and associated metatranscriptome reads were obtained from each enrichment culture.To avoid biases in estimating gene expression, we selected seven high-quality MAGs representing the dominant taxa from each enrichment culture as reference genomes.High numbers of transcript read obtained from all of these enrichment cultures mapped to the key genes in Phoenicimicrobium oleiphilum for n-alkanes degradation, RGP, acetogenesis, and hydrogenases, suggesting an active role in the degradation of a variety of n-alkanes and producing H 2 , CO 2 , and acetate (Figure S15).These results extend the role of JS1 to the fermentation of larger n-alkanes rather than only short-chain n-alkanes, which was suggested previously [1].The expression of these genes in samples from the Haoxian Oil Reservoir and these enrichment cultures imply a role for Phoenicimicrobium oleiphilum in the fermentation of hexadecane, eicosane, and triacontane to H 2 , CO 2, and acetate.

Metabolic handoffs in a thermophilic acetate-oxidizing enrichment
To better understand the ecology of Thermatribacter velox B11 T in the acetate-amended enrichment culture from which it was isolated, we examined the metagenome of the culture and considered syntrophy and metabolic handoffs [60][61][62][63], noting that very little is known about anaerobic processing of acetate at high temperatures (75 ºC).Only a single MAG encoded the complete gene set for methyl coenzyme M reductase, mcrABCDG, the hydrogenotrophic methanogen Methanothermobacter [58] (Table S3), whose relative abundance increased from 6.53 (HX-OS) to 15.49% (HX-AS) after acetate Fig. metatranscriptomic of oleiphilum from crude oil-, hexadecane-, eicosane-, and triacontane-enriched samples.The laboratory enrichments were performed in anoxic PRM at 55 °C with oily sludge as inoculum.The supplementary substrates were added by 1 ml/L hexadecane, 2 ml/L eicosane, 0.6 g/L triacontane, and 200 g/L oily sludge, respectively enrichment.The complete gene sets for both WLP and RGP were detected in the metagenome, suggesting that two separate syntrophic acetate degradation pathways (SADPs) might operate, with a single methanogen as a hydrogen sink (Fig. 8b) [64].The relative abundance of Bacillota (Firmicutes) increased from 0.01 (HX-OS) to 6.55% (HX-AS) after acetate enrichment.These Bacillota included the genus Thermoanaerobacter and uncultivated members of the Moorellales, and most encoded the potential for complete oxidation of acetate to H 2 and CO 2 via the WLP [64].Although more than a quarter of the Bacillota cells were replicating based on bias in the number of reads near the origin of replication (iRep > 1.25; Table S3) [34], the fastest replicating organism was Thermatribacter velox B11 T (iRep > 2).Considering B11 T was unable to grow as a pure culture with acetate alone and the presence of the RGP and hydrogenases, we speculate that it may participate in the partially syntrophic oxidation of acetate.Given its fast replication rate, we propose that the RGP-based SADP may compete with the WLPbased SADP under these conditions.Other organisms in the acetate enrichment may provide common goods for acetate oxidation or may consume metabolic intermediates or necromass.For example, only a single MAG assigned to the genus Thermococcus_A encodes the key genes necessary for serine biosynthesis, using a single enzyme with both D-serine dehydratase and racemase domains (Figure S26).Several other MAGs encoded genes for formate dehydrogenase (Table S3), suggesting formate may be an important metabolic handoff, which is in line with similar findings from other hydrothermal environments [63].

Conclusions
Atribacterota is a globally distributed phylum but previously only one pure culture, Atribacter laminatus RT761 T , and two described species from genomic assemblies Ca.Caldatribacterium saccharofermentans and Ca.Caldatribacterium californiense have been described [2,6].Here, we isolated a second member of the phylum from a terrestrial oil reservoir, Thermatribacter velox B11 T , which is the first pure culture in the newly described family Thermatribacteraceae.This species was capable of fermenting a variety of carbohydrates, particularly d-mannose, d-fructose, d-ribose, d-xylose, d-galactose, d-trehalose, and xylan, with subsequent formation of acetate, ethanol, H 2, and CO 2 as end products, broadening the scope of carbohydrate fermentation within the class Atribacteria.Yet, given its enrichment in acetatefed cultures, and gene presence of hydrogenases and partially RGP in B11 T , we speculate that B11 T and likely many other Atribacteria may participate in the partially RGP-based SADP.This metabolic flexibility may allow members of the Atribacteria to alternatively ferment either primary biomass substrates (i.e., poly-, oligo-, and monosaccharides) when available or simple substrates produced geochemically or through fermentation, which may be more reliable in the anoxic, carbon-rich environments where Atribacteria thrive.
A different species of Atribacterota, herein named Phoenicimicrobium oleiphilum HX-OS.bin.34TS , was more abundant than Thermatribacter velox B11 T in the natural oil reservoir samples.We noted that Phoenicimicrobium oleiphilum and several other genomes from class JS1, herein named Phoenicimicrobiia, encode fumarate-adding enzymes.These genes, together with mcmLS, NiFe hydrogenase Group 4b, and FeFe hydrogenase Group A3 strongly suggest a role in alkane degradation.In support of this, we were able to cultivate Phoenicimicrobium oleiphilum with crude oil or a variety of n-alkanes and demonstrate the presence of transcripts in these cultures mapping to key metabolic genes, including faeABCDEF, RGP, and hydrogenases.Phylogenetic analyses and synteny of faeABCDEF subunits suggested that anaerobic hydrocarbon degradation was ancestral and may be widespread in Phoenicimicrobiia.Furthermore, we also note the absence of the WLP in Phoenicimicrobiia, but note the universal presence of the RGP as a metabolic hub for both acetate oxidation and CO 2 /formate assimilation.The phylogenies of hydrogenases, CODH/ACS complex, RGP, and FaeA are overall congruent with the phylogenetic divergence of Atribacteria and Phoenicimicrobiia, indicating an apparent lateral gene transfer after their divergence and vertical inheritance in Atribacteria and Phoenicimicrobiia respectively.The reconstruction of the metabolic networks revealed the likely roles of distinct members of the Atribacterota in hydrocarbon-and acetate-rich environments within syntrophic metabolic networks.We believe that these novel insights into the diversity, physiology, ecology, and evolution of Atribacterota will contribute to further laboratory cultivation and promote more incisive studies of their syntrophic biology in other habitats worldwide.
The type strain B11 T (=CCAM 969 T =JCM 39351 T ) was isolated from an oil sludge collected from an oil tank of Shengli Oilfield in China.
The description is the same as that of the genus Thermatribacter.
Type genus: Thermatribacter.The description of the class is the same as for the order Phoenicimicrobiales.

Fig. 3
Fig. 3 Carbohydrate fermentation and products of Thermatribacter velox B11 T .a Volatile fatty acids and ethanol produced from saccharides and the polysaccharide xylan after 142 h incubation.Control, no extra substrate.b Fatty acids and ethanol production during fermentation of glucose.c Hydrogen and carbon dioxide production during fermentation of glucose

Fig. 8
Fig. 8 Metabolic models in the hydrocarbon-and acetate-enriched environments.The metabolic models are based on the 33 representative genomes.a Genome and metatranscriptome resolved model in the hydrocarbon-enriched environments.b Metagenome and genome resolved model in the acetate-enriched environments